Fig. 2 |. Mouse exT_reg cell classifier genes identify human exT_reg cell candidate genes and surface markers in a human scRNA-seq and CITE-seq dataset of CD4⁺T cells.

a, Differentially expressed mouse exT_reg classifier genes were examined among all CD4⁺ T cell clusters from a published single-cell human dataset from 61 men and women (aged 40–80 years). Statistical significance for the enrichment of each gene in one cluster versus all others was assessed. Average log₂ fold change (log₂FC, dot color) and positive cell proportions (pct.1, dot size) for significantly (P < 0.05) enriched genes are shown. Six highly expressed exT_reg genes (CST7, NKG7, GZMA, PRF1, TBX21 and CCL4) enriched in human cluster CD4T_7 (red boxes). b, Feature plot showing expression of FOXP3 gene (blue dots) in the human CD4⁺ T single-cell dataset. T_reg cell cluster CD4T_17, previously identified as T_reg cells, highlighted. c, Frequencies of cells that expressed each of the exT_reg cell signature genes in CD4T_7 (red circles) and CD4T_17 (T_reg cells, blue circles). n = 61. d, Cells expressing optimal exT_reg cell candidate gene combination (CST7 + NKG7 + GZMA + PRF1 + TBX21) are highlighted in red on UMAP embeddings of CD4⁺ T clusters from the scRNA-seq data. All other cells are in gray. e, Differentially expressed surface markers (CITE-seq antibodies) on cells expressing candidate gene combinations. Average log₂FC (dot color) and log₂(pct.1/pct.₂) × avg_log₂FC (dot size) for significant (P < 0.05) differentially expressed protein markers on candidate cells versus CD4T_17 (T_reg cells) are shown. Enriched exT_reg markers CD127, CD16 and CD56 are marked with red boxes. The second-to-last combination is candidate 5 (CST7 + NKG7 + GZMA + PRF1 + TBX21). f, UMAP embeddings of CD4⁺ T clusters. Black outline marks cluster CD4T_7; cells that coexpressed CD16 and CD56 are shown as red dots. g, Representative plots from flow cytometry (FACS) showing CD16⁺CD56⁺CD4⁺T cells (exT_reg cells, red) and CD25⁺CD127^loCD4⁺T cells (T_reg cells, blue) with FMO controls. h, MFI of CD16 (n = 6), CD56 (n = 6), CD127 (n = 3) and FOXP3 (n = 6) expression in exT_reg cells (red circles) and T_reg cells (blue circles). 20% male, 80% female donors; ages 23–64 years. Results (c and h) are represented as the mean ± s.e.m. Each dot (c and h) represents a biological replicate from an independent human donor. Statistical comparisons using a two-tailed Mann–Whitney U test (c and h) and a two-tailed Wilcoxon’s rank-sum test with Benjamini–Hochberg correction for multiple comparisons (a and e). In c, ****P < 0.000000000000001 for CST7, NKG7, GZMA, PRF1 and ****P = 3.86 × 10⁻¹³ for TBX21. In h, *P = 0.0121, **P = 0.0022.